One of the most important economic goals in the poultry industry is to minimize losses caused by infectious diseases by means of effective vaccines. Among the prominent virus pests are avian adenoviruses which cause various diseases in poultry. Adenoviruses are double-stranded DNA viruses with a genome in the range of 25-45 kb [Jucker et al., J. General. Virol. 77:469-479 (1996)]. The virus consists of 11 proteins with molecular weights ranging from 14kD to 97kD [Nazerian, K., et al., Avian Dis. 35:572-578 (1991)]. The polypeptide 97kD, hexon, is a monomer of the major outer capsid. Other capsid proteins include the structural protein penton, and fiber proteins which are involved in the interaction with cell receptors during virus penetration into the cell.
One of the main diseases in turkeys is caused by the avian adenovirus Hemorrhagic Enteritis virus(HEV). The virus suppresses elements of the immune system by destroying B cells and macrophages. As a result, the immune response to diseases is decreased, as well as the effectiveness of response to various vaccines. The disease is especially prevalent during the ages 7-9 weeks [Domermuth, C. H. and Gross, W. B., 1984 Diseases of poultry, Iowa State University press, Ames, Iowa, 8th Edition (1984) pp. 511-516] when the birds are no longer protected by maternal antibodies [Van den Hurk, J. V., Avian Dis. 30:662-671 (1986); Harris, J. R., and Domermuth, C. H., Avian Dis. 21:120-122 (1977); Fadly, A. M., and Nazerian K., Avian Dis., 33:778-786 (1989)]. Heavy financial losses result from outbreaks of HEV, with its symptoms of weight loss and mortality. Furthermore, outbreaks of other diseases can follow as result of lowered resistance.
Another poultry disease caused by an adenovirus, is the egg drop syndrome. The virus was detected in 1976 [Van Eck, J. H. H. F. G. Davelaar, T. A. M., Avian Pathol., 5:261-272 (1976)], and is designated as strain 127 [McCracken, R. M. and McFerran, J. B., Avian Pathol, 7:483-490 (1978)]. The Egg Drop Syndrome virus leads to creation of eggs having a thinner shell or to shell-less eggs. These phenomena cause a significant reduction in egg production throughout the world and therefore lead to economic losses [McFerran, J. B., Rowley, H. M., McNulty, M. S., and Montgomery, L. J., Avian Pathol., 6:405-413 (1997)]. The virus is transmitted through direct or indirect contact with infected fowl. The main way the virus spreads is by vertical transmission, from hen to eggs, which can be prevented only by keeping uninfected flocks as a source for further breeding. There is no successful treatment of the disease.
A conventional vaccination against HEV is made either by inactivated virus, or by a live vaccine of low virulence, for example by an attenuated virus [Fadly and Nazerian (1989)]. The live vaccines may show a disadvantage of antigenic coverage not identical to the virulent strain. On the other hand, the commercially available inactivated vaccines may expose the animals to an unnecessary wide range of antigens. Another vaccination against EDS has been also described [Baxendale, W., Lutticken, D., Hein, R., and McPherson, I., Avia Pathol., 9:77-91 (1980); Solyom, F., Nemesi, M., Forgacs, A., Balla, E., and Perenyi, T., Dev. Biol. Stand., 51:105-121 (1982)]. It is performed at the age 14-16 weeks by inactivated vaccine in oil adjuvant, and it confers one-year protection against clinical symptoms. The use of attenuated vaccines, and even inactivated ones, always bears a danger due to the possibility that the inactivation may be incomplete, or that the mild, attenuated virus will revert to virulence.
It is therefore an object of this invention to provide a safe vaccine against HEV and against EDS that eliminates the danger of an outbreak of the disease which is inherent to the use of live or inactivated vaccines, as a result of reversion of the virus to virulence, or its incomplete inactivation. It is a further object of this invention to protect poultry against a wide antigenic range of the virulent field viruses. Another purpose of this invention is to prevent unnecessary exposure of the birds to non-relevant antigens during the vaccination.
There is an additional problem in a conventional vaccination against HEV; the heavy expenditure involved in relying on spleen tissue from live turkeys for propagation of the virus in large quantities. The inactivated vaccines used at present against HEV are expensive and involve mass infection of birds in order to isolate the virus from the spleen.
Recombinant technology enabled construction of new vaccines. Efficacy of immunization by the recombinant virus has been reported for a number of disease-causing viruses. The infection of birds by the recombinant virus, that expresses the protein haemaglutinin of Avian Influenza, provided protection to birds exposed to this disease [Taylor. J., et al., Vaccine 6:504-508 (1988); Tripathy, D. N., and Wittek. R., Avian Dis. 34:218-220 (1990)]. The immunization of birds with recombinant FPV, into which the gene of the HN protein of the Newcastle Disease Virus (NDV) had been inserted, induced production of antibodies which protected them against the disease (Edbauer C., et al., Virology, 179:901-904 (1990)]. Furthermore, it was found that the birds which had been immunized with this recombinant virus developed resistance to NDV without any decrease in the resistance to FPV [Ogawa, R., et al., Vaccine 8: 486-490 (1990)]. Recombinants of this sort are effective for the delivery of vaccines to birds [Schnitzlein W. M., et al., Virus Res. 10: 65-76 (1988)]. Evidently, protecting against two diseases by one vaccine makes vaccination considerably cheaper.
In developing a poultry vaccine, a number of factors concerning the poultry industry must be taken into account. Since the price of a single bird is relatively very low, one of the most important factors is the cost of the vaccine. A vaccination by a subunit viral protein might be an inexpensive approach. This approach would also eliminate the said danger of reversion to virulence, since a subunit vaccine cannot replicate. Another advantage would be elimination of unnecessary exposure to a variety of antigens. The effectiveness of subunit viral vaccines has been tested in the inventors' laboratory on Infectious Bursal Disease virus, whose VP2 was expressed in baculovirus expression system, and it conferred full protection against the disease [Pitcovski. J., et al., Avian Dis. 40:753-761 (1996)]. Other subunit vaccines have been found efficient, including vaccine against hepatitis B in humans.
Subunit vaccine against EDS virus based on the fiber protein, have been previously reported by the inventors Fingerut, E., B. Gutter, G. Gallili, A. Michael, and J. Pitcovski. 2001. Development of a subunit vaccine to EDS. In: XXXVIII Ann. Conv. World Poultry Sci. Assoc. (Hebrew).
In this publication, the present inventors describe creation of recombinant EDS fiber protein and use thereof as vaccine against EDS. However, as shown by the following Examples, and particularly in Example 5 the vaccine of the present invention which is based on a specific fragment of the fiber protein, is significantly more efficient as an anti-EDS vaccine (33% vs. 100% virus neutralization). The Fiber fragment used in the present invention comprises the knob domain and part of the N′ terminal shaft domain of the fiber protein. Interestingly, use of the corresponding fragment in another avian adenovirus, HEV as exemplified in the present invention (Examples 1 to 3), efficiently protects treated birds against HEV. Therefore, the particular use of certain fragments of the fiber protein in the recombinant protein of the invention leads to development of an efficient anti-adenovirus vaccine.
It is therefore an object of the present invention to provide subunit vaccines against adenoviruses. Such vaccinating compositions comprise as an active ingredient recombinant protein comprising a fragment of the fiber protein of any adenovirus. This specific recombinant protein is capable of eliciting a protective immunity in any infected animal, against said adenoviral pathogen. More specifically, the recombinant protein of the invention is intended for vaccinating human or any domestic animal against adenoviral infection. For example, particular object of this invention is to protect poultry against two economically main diseases, HEV and EDS, by producing immunogenic proteins of both EDS virus and HE virus as subunit vaccines.